Malaria remains a major cause of illness and death worldwide. Two major unresolved issues concerning the pathogenesis of malaria are: 1) what molecules from the parasite activate the innate immune system and cause inflammation and, 2) what are the receptors that are ligated by these microbial products. The overall goal of this project is to identify the molecular mechanisms of systemic inflammation and fever in individuals infected with Plasmodium falciparum. Our preliminary data demonstrate that malaria DNA, especially when complexed to the malarial pigment hemozoin, activates inflammation. The underlying HYPOTHESIS of this proposal is that fever and inflammation in acute malaria are initiated by the interaction of malarial DNA with DNA receptors expressed by phagocytes. We further hypothesize that the innate immune response to malarial DNA is mediated by two receptors: TLR9 and an as yet unidentified receptor for AT-rich DNA motifs. This hypothesis challenges the concept that malarial glycosylphosphatidylinositol (GPI)-lipid anchors, which are primarily TLR2/CD14 agonists, are the primary cause of systemic inflammation in malaria. In order to test this hypothesis, we propose to combine an examination of immune activation in cells from patients with acute febrile malaria with in vitro work. We propose three Aims. The first Aim is to determine if innate immune activation induced by malaria, occurs primarily in TLR9+ cells, or in cells that primarily express TLR2 and/or CD14. We will profile gene induction in purified cells with defined TLR expression in culture and from patients in order to determine which receptors and signaling pathways are employed. DNA, and oligonucleotides derived from the malarial genome, will be compared to purified malarial GPI anchors. In our second Aim, we will assess the importance of hemozoin in inflammation. PBMC will be collected from malaria patients, sorted into hemozoin positive and negative monocytes and DCs, and assessed for markers of immune activation. We will perform confocal imaging studies of hemozoin and malarial DNA trafficking in immune cells. These studies will determine the temperospatial relationships between trafficking and innate immune responses, including the engagement of specific DNA receptors and the activation of the inflammasome. In the final Aim, we will focus on the immune activity of AT-rich oligonucleotides whose sequences are derived from the malarial genome. We will focus on; 1) those signaling pathways that result in the production of Type I interferons, which we have found to be a critical determinant of outcome in mouse cerebral malaria; and 2) activation of the inflammasome, primarily through NRLP3 and AIM2, an inflammasome component that we recently discovered. Signaling pathways will be established using both a molecular genetics approach and the use of proteomics. We will test inflammasome knockout mice, including a new AIM2 KO, in mouse cerebral malaria, thus relating our in vitro findings with in vivo disease. Our ultimate goal is to define the relative role of malarial DNA in mediating innate immune responses during malarial infection and to definitively identify related DNA receptors. PUBLIC HEALTH RELEVANCE: Malaria is the world's most common infectious disease, and kills millions of individuals annually. US citizens risk obtaining malaria when they travel or are engaged in military operations in tropical areas. The purpose of this grant is to gain a better understanding of why malaria causes disease in the hopes that better therapies can be devised, including an effective vaccine that might prevent malaria.